European Journal of Pharmacology, 70 (1981) 589--590 © Elsevier/North-Holland Biomedical Press Rapid communication SOLUBILIZATION OF RAT BRAIN a l-ADRENOCEPTORS PASCALE GUICHENEY, ALAIN RAPPAPORT and PHILIPPE MEYER * Physiologie et Pharmacologie Vascuiaire et Rdnale, INSERM U7, CNRS LA 318, H6pital NECKER, 161 rue de Sdvres, 75015 Paris, France Received 26 February 1981, accepted 4 March 1981 589 a-Adrenergic receptors are classified into two subtypes al and a2 according to their affinity for various agonists and antagonists (Starke et al., 1979). The two classes are pre- sent in rat brain (Greenberg et al., 1976; Miach et al., 1978). In order to know if the two a-adrenocep- tors correspond to different molecular com- plexes, detergent solubilization was carried out on rat brain membranes. Successful results were obtained with a l-adrenoceptors after prelabelling with the specific a ~-adrener- gic ligand [3H]prazosin. Preliminary results are reported in the present communication. Fresh brain tissue obtained from male Wistar rats was homogenized in 0.32M sucrose and submitted to a 1200 × g centrifu- gation for 10 min at +4°C. The resulting supernatant was centrifuged at 30 000 × g for 20 min at +4°C. The membrane pellet was then resuspended in 50 mM Tris-HC1 pH 7.6 and centrifuged 10 min at 49 000 × g (+4°C). After resuspension in the same buffer, mem- branes were incubated at 25°C for 30 min with [3H]prazosin 2nM, a concentration which was shown to ensure a maximal binding in intact brain membranes (Miach et al., 1980). As previously reported for hepatic mem- branes (Guellaen et al., 1979), prelabelling of binding sites with the tritiated antagonist prior to the treatment with detergent appeared necessary for a satisfactory recov- ery. * To whom correspondence should be addressed. The prelabeUed [3H]prazosin binding sites were thus solubilized by treatment with 0.3% deoxycholate at +4°C during 30 min, fol- lowed by centrifugation at 125 000 × g for 1 h. Non-ionic detergents, such as lubrol PX, and Triton X-100 were ineffective at various concentrations. The soluble [3H]prazosin-binding protein was assayed using two procedures. (i) Gel filtration on Sephadex G50 medium column (27 × 0.5 cm), equilibrated and run with 50 mM Tris-HC1, 0.3% deoxycholate pH 7.6. Fractions of the effluent (300 gl) were col- lected in Unisolve and counted in a liquid scintillation spectrometer. Bound [3H]prazo- sin appeared as a radioactive peak in the void volume. Preincubation of membranes before deoxycholate treatment with an excess of un- labelled prazosin and phentolamine inhibited the binding in a concentration-dependent fashion. 10 -7 M of these compounds resulted in the complete supression of the excluded radioactivity. (ii)Solubilized proteins of the 125 000 × g supernatant (150 #1) were precip- itated by 1.2 ml of 10% polyethylene/glycol 6000 (PEG) in 50 mM Tris-HC1 pH 7.6, in the presence of 500 mg of 7-globulin. The reSult- ing precipitate, stable at +4°C for up to 30 min, was filtered on Whatman GF/F glass fiber filters and rinsed with 6 ml of 8% PEG buffer. Non-specific binding determined in the presence of 10-SM phentolamine repre- sented 20% of the total radioactivity. Deoxycholate concentration 0.3% ensured the recovem.T of 50--75% of the [3H]prazosin binding site,:. Dresent on the crude membrane